Abstract Ewing sarcoma (ES) is a rare but deadly bone tumor which occurs mainly in adolescents and young adults.1 As survival for ES has not improved beyond ~60% in the last few decades, it is critical to enable the prediction and early detection of disease through the discovery of genetic contributions to risk. While the literature on the causes of ES is sparse, several lines of evidence have emerged which collectively suggest that de novo germline mutations (DNM's) underlie a proportion of cases. These include a lack of environmental risk factors or syndromes associated with ES and a strong increasing risk with rising parental age. Given the strong rationale for examining DNMs in ES, we propose the following specific aims using WGS data of germline from 331 case-parent trios: 1) to identify germline DNMs in ES patient genomes; 2) to identify regions with recurrent DNMs in ES patient genomes; and 3) to compare the burden of pathogenic variation from DNMs or otherwise in an extended set of 457 ES patients with WGS data to controls. The analysis will be the definitive examination of DNMs in ES. Results indicating DNMs are a frequent cause of ES could lead to clinical guidelines urging sequencing of all patients, who currently receive little genetic testing due to lack of family history. Lastly the results could potentially point to opportunities for early detection and possibly therapeutic targets.